CN1283790C - Carrier material in use for preparing dextran through method of enzyme immobilization - Google Patents
Carrier material in use for preparing dextran through method of enzyme immobilization Download PDFInfo
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- CN1283790C CN1283790C CN 200410014621 CN200410014621A CN1283790C CN 1283790 C CN1283790 C CN 1283790C CN 200410014621 CN200410014621 CN 200410014621 CN 200410014621 A CN200410014621 A CN 200410014621A CN 1283790 C CN1283790 C CN 1283790C
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- dextran
- water
- soluble
- alginic acid
- soluble polymer
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Abstract
The present invention relates to a carrier material for preparing dextran by an enzyme immobilization method. A compounded macromolecule gel which is composed of algae acid or alginate and a natural and/or synthetic water soluble or semi-water soluble macromolecule is used as the immobilized carrier material to immobilize dextransucrases and produce the dextran. The carrier material has the advantages of high intensity, firm adherence and repeated use, obviously increases the output and the quality of dextran, and is favorable to the process control of industrial production.
Description
One, technical field
The present invention relates to the solid support material in a kind of material in the biological enzyme technology, particularly immobilized enzyme method technology, exactly is that a kind of enzyme immobilization method is produced the solid support material that dextran is used.
Two, background technology
Dextran (Dextran) has another name called dextran, is a kind of good plasma substitute, has effects such as antithrombotic, microcirculation improvement, and has been widely used in fields such as medical treatment, foodstuffs industry, biology.
What the traditional production technique of dextran adopted is fermentation technique, " preparation of dextran and application " (" Sichuan chemical industry and corrosion control " 2001 as Zhang Chaohui, 4 (1), 50-52), people such as Mariana Santos have also used fermentation technique (" Biochemical Engineering Journal " 2000 when the research dextran is produced, (4), 177-188).Though this Technology is comparatively ripe, the ethanol consumption is big in producing, and production environment is abominable, and impurity such as the nitrogen in the product, chlorine or ion are difficult to control, and molecular weight distribution is inhomogeneous, and the later stage separates, purification ratio difficulty, causes its quality product lower.People such as Wang Yong are in " dextran is produced novel process research " (" biotechnology " 1994,4 (3), adopted the orientation fermentation technology to realize the stage treatment of dextran molecule amount in 45-46), avoided the acid degradation of use salt, reduce impurity such as chlorion, and reduced the alcoholic acid usage quantity.But thalline and dextran twine mutually in process of production, can not separate, and cause impurity such as containing foreign protein in the product.Adopted membrane separation technique in the disclosed dextran production technique of CN1415631A, although this technology has been avoided the use of alcohol and some organic solvents, but Pyatyi ultrafiltration membrance filter, separating treatment, efficient is not high in industrial production, and exists a series of problems such as concentration polarization and fenestra pollution in the membrane technique method.Other classification, separation method or technology of dextran, carry out dextran molecule amount fractionated research (" Journal of Chemical Technology andBiotechnology " 1989 as human chromatographic columns such as Barker P.E., 46 (3), 209-218) etc., all be that a certain step of traditional zymotic production technique is carried out some technique variation or processing, and can not make the improvement or the raising of essence.
What the disclosed dextran production technique of CN1353193A adopted is the enzyme process technology.Biological enzyme technology is for traditional fermentation technique, improved the output of dextran, but produced dextran, still will use hydrochloric acid in process of production with resolvase, still keep away the introducing of foreign ions such as unavoidable chlorine, some impurity such as while foreign protein still are difficult to remove.When people such as Monsan produce at the research dextran, investigate the enzyme work of having compared resolvase and immobilized enzyme and wait some features (141-157), immobilized enzyme is better than resolvase for " Methods in Enzymology " 1987, (136).M.M.Abdel-Halimd, people such as El-Sayed are producing dextran (" Biotechnologyand Bioengineering " 1990 with fed-batch in batches, 36 (6), 338-345) and at semicontinuous batch feeding produce dextran and dextransucrase (" Biotechnology and Bioengineering " 1990,36 (6), be that Leuconostoc mesenteroides is fixed on the alginate calcium in research 346-351).N.V.Sankpal, people such as A.P.Joshi are producing with immobilization technology in the research of dextran, rhizopus is fixed on the porous cellulose (" ProcessBiochemistry " 2001,37 (4), 395-403).Serve as the fixation support material with alginates, the pore of gel parent is easily stopped up by dextran, reduced substance transportation, and the gel-strength of alginates is not high, the alginates gel particles of immobilized enzyme is broken easily, life cycle is shorter, thereby has influenced the control of ultimate capacity and the quality and the production process of dextran.With fixing of porous cellulose solid support material, overcome some shortcomings of alginates, but fixation rate is not high, the ultimate capacity of dextran and quality still are undesirable.
The suitability for industrialized production dextran, enzyme process is better than fermentation method, and immobilized enzyme method is better than free enzyme process.Solid support material is the important factor that influences immobilized enzyme method technology.
Three, summary of the invention
The present invention is for fear of the defective of above-mentioned various production methods and technology, improves the output and the quality of dextran, adopts natural and/or synthetic water
The plural gel that dissolubility or half water miscible polymer and alginic acid and/or alginic acid salt constitute serves as the fixation support material and fixes dextransucrase, and is used for producing dextran.It is characterized in that each component has following weight percent:
Alginic acid and/or alginic acid salt 55～99%
Natural water-soluble and/or half water-soluble polymer 0～25%
Synthetic water-soluble and/or half water-soluble polymer 0～20%
Two kinds of macromolecular materials are not zero simultaneously.
Optimum ratio:
Alginic acid and/or alginic acid salt 60～95%
Natural water-soluble and/or half water-soluble polymer 1～20%
Synthetic water-soluble and/or half water-soluble polymer 1.5～20%.
Optimal components ratio:
Alginic acid and/or alginic acid salt 75～90%
Natural water-soluble and/or half water-soluble polymer 4～15%
Synthetic water-soluble and/or half water-soluble polymer 3～10%.
Described natural water-soluble or half water-soluble high-molecular material is selected from one or more in gelatin, chitosan and the carrageenin.
Described synthetic water-soluble or half water-soluble high-molecular material is selected from one or more among sodium polyacrylate, sodium polymethacrylate, polyvinyl alcohol and the PVP.
Immobilization and immobilized enzyme operation process are as follows:
Get above-mentioned polymer composite carrier and add the gelating soln that water is made into 2～10% concentration, in this gelating soln, add a certain amount of bacterial suspension nutrient solution and mixing, splash into calcium chloride (CaCl then
2) form dripping pill in the solution, and allow its cross moulding 1～2 hour.Separate sterilized water washing dripping pill.This dripping pill set has the bacterium through suspension culture.
Under aseptic condition dripping pill is inserted in the inducing culture, the constant temperature shaking table is cultivated, and induces the generation biological enzyme.Reaction finishes after-filtration and separates, and washs dripping pill with sterilized water.This dripping pill set has dextransucrase, i.e. immobilized enzyme.
Generate the high-molecular weight dextran with carrying out enzymatic conversion reaction in the pure sucrose solution of immobilized enzyme dripping pill access substrate.The enzymatic conversion reaction after-filtration separates each time, and dripping pill is cleaned with sterilized water, inserts the next batch substrate again and reuses.Filtrate obtains dextran through the ethanol precipitating.
The present invention has fundamentally avoided foreign ions such as nitrogen that traditional technology and some other Technology brought in process of production, chlorine, has reduced the alcoholic acid usage quantity.Simultaneously, use the polymer composite gel material to serve as fixation support, gel-strength and action effect behind the enzyme immobilization strengthen greatly, but the repeating utilization factor of immobilized enzyme and dripping pill improves, life cycle prolongs, the output and the quality of dextran obviously improve, and help the control of actual production operation and production process.This enzyme immobilization technology provides strong technical support for easy, quick, High-efficient Production dextran.
Compared with prior art, the present invention has the following advantages:
1, advanced technology
The synthetic dextran of the biological enzyme that comes out with culture of isolated can be eliminated impurity or ions such as the nitrogen that brings in the production process, chlorine.
2, production cost reduces
The utilization enzyme immobilization technology can make the production process serialization, and enzyme can reuse, and production cost is significantly reduced.
3, quality product improves, and output increases
Utilize the enzyme engineering technology transformation to have industry now, its technology upgrading is regenerated, thereby the foreign matter content of control product makes product molecular weight distribution even, quality improves.
Four, embodiment
(1), the preparation of polymer composite carrier
1, get 99 parts of alginic acid or alginic acid salt, 1 part of gelatin or PVP mix.
2, get 75 parts of alginic acids, 15 parts in gelatin, 10 parts of sodium polyacrylates mix.
3, get 60 parts of alginic acid salt, 15 parts of carrageenins, 10 parts in gelatin, 10 parts of sodium polyacrylates, 5 parts of PVP mix.
4, get 50 parts of alginic acids, 40 parts of alginic acid salt, 5 parts of chitosans, 5 parts of polyvinyl alcohol mix.
5, get 30 parts of alginic acids, 25 parts of alginic acid salt, 10 parts in gelatin, 15 parts of chitosans, 10 parts of sodium polymethacrylates, 10 parts of PVP mix.
6, get each 40 parts of alginic acid, alginic acid salt, respectively 5 parts of gelatin, carrageenin, chitosan, polyvinyl alcohol mix.
7, get each 35 parts of alginic acid, alginic acid salt, 10 parts in gelatin, 9 parts of polyvinyl alcohol, 6 parts of sodium polyacrylates, 5 parts of PVP mix.
(2), the cultivation of bacterium
With the Leuconostoc mesenteroides is example.
Get Leuconostoc mesenteroides L.mesenteroides--1226 through culture dish solid culture, fluid suspension culture.
The prescription of substratum:
Liquid nutrient medium: sucrose≤20%, peptone≤0.2% and Na
2HPO
4≤ 0.2%.
Solid medium: sucrose≤20%, peptone≤0.2%, Na
2HPO
4≤ 0.2% and agar 2%.
It is standby to get the bacterial suspension nutrient solution at last.
(3), enzyme is fixed
Get the bacterial suspension nutrient solution of 25ml, add 75ml and add mixing in 5% the gelating soln of water preparation by the solid support material of (one) preparation, splash into CaCl then
2Form dripping pill in the solution, and allow its crosslinked 1～2 hour.
Dripping pill is cleaned with sterilized water, under aseptic condition, be linked on the enzyme induction substratum.At rotating speed≤200rpm, cultivate the generation of inducible enzyme on the constant temperature shaking table that temperature is 20～30 ℃.After 48 hours, reaction system is filtered, with sterilized water Xian Di dripping pill, being fixed enzyme.
Enzyme induction substratum: sucrose≤20%, peptone≤0.2%, Na
2HPO
4≤ 0.2%, MnSO
47H
2O≤1% and CaCO
3≤ 15%.
(4), produce dextran
The immobilized enzyme dripping pill is inserted sugar degree less than in 15% the pure sucrose solution, and a large amount of dextrans generate after 48 hours.Separate, filtrate promptly is dextran with 95% ethanol precipitating, precipitating thing.The immobilized enzyme dripping pill is reused.
Claims (5)
1, a kind of enzyme immobilization method is produced the solid support material that dextran is used, and it is characterized in that having following component and weight percent:
Alginic acid and/or alginic acid salt 55～99%
Natural water-soluble and/or half water-soluble polymer 0～25%
Synthetic water-soluble and/or half water-soluble polymer 0～20%
Two kinds of macromolecular materials are not zero simultaneously.
2, solid support material according to claim 1 is characterized in that:
Alginic acid and/or alginic acid salt 60～95%
Natural water-soluble and/or half water-soluble polymer 1～20%
Synthetic water-soluble and/or half water-soluble polymer 1.5～20%.
3, solid support material according to claim 1 and 2 is characterized in that:
Alginic acid and/or alginic acid salt 75～90%
Natural water-soluble and/or half water-soluble polymer 4～15%
Synthetic water-soluble and/or half water-soluble polymer 3～10%.
4, solid support material according to claim 1 is characterized in that: described natural water-soluble or half water-soluble polymer is selected from one or more in gelatin, chitosan, the carrageenin.
5, solid support material according to claim 1 is characterized in that: described synthetic water-soluble and/or half water-soluble polymer is selected from one or more among sodium polyacrylate, sodium polymethacrylate, polyvinyl alcohol, the PVP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410014621 CN1283790C (en) | 2004-04-08 | 2004-04-08 | Carrier material in use for preparing dextran through method of enzyme immobilization |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410014621 CN1283790C (en) | 2004-04-08 | 2004-04-08 | Carrier material in use for preparing dextran through method of enzyme immobilization |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563374A CN1563374A (en) | 2005-01-12 |
| CN1283790C true CN1283790C (en) | 2006-11-08 |
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| CN 200410014621 Expired - Fee Related CN1283790C (en) | 2004-04-08 | 2004-04-08 | Carrier material in use for preparing dextran through method of enzyme immobilization |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100417724C (en) * | 2006-07-14 | 2008-09-10 | 清华大学 | Nanometer carbonic anhydrase grain for biological catalysis of polymer and its prepn process |
| CN102220244B (en) * | 2011-05-06 | 2013-01-09 | 合肥工业大学 | Hypocrea lixii strain and method for preparing dextran enzyme with the Hypocrea lixii strain |
| CN103627692B (en) * | 2013-11-13 | 2015-12-09 | 华南理工大学 | Utilize the method for using modified bagasse immobilization carbonic anhydrase |
| WO2016200773A1 (en) * | 2015-06-10 | 2016-12-15 | Vuv Analytics, Inc. | Method for detailed and bulk classification analysis of complex samples using vacuum ultra-violet spectroscopy and gas chromatography |
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